The calcium-activated chloride channel (CaCC) was first observed in salamander retina in the early 1980s and has since then been found to be responsible for diverse phenomena ranging from signal transduction in the olfactory sensory neuron to fluid secretion in the epithelium. In disease, the CaCC has been considered a target for the treatment of hypertension, cystic fibrosis, and cancer. Despite its importance, the gene encoding the CaCC was not identified until 2008 when three laboratories independently discovered a TMEM16A clone that produced the CaCC current. Building on this discovery, the project described in this research plan will begin the initial basic biophysical characterization of this channel by determining the number of TMEM16A subunits that oligomerize to form a functional CaCC. Preliminary data indicate that TMEM16A co-immunoprecipitates specifically with other TMEM16A molecules, suggesting that more than one subunit is present in the CaCC complex. To determine the exact number of subunits, TMEM16A will be linked to two different tags and heterologously expressed. Using electrophysiology, immunoprecipitation, and biotinylation, the proportion of CaCC complexes that contain only subunits with one tag but not the other will be observed. This proportion is a function of the number of subunits required to assemble a channel - the more subunits required, the less likely that they are all the same - and is modeled by the bionomial distribution. To validate the results from this approach, the apparent molecular weight of TMEM16A-containing CaCC complexes under native and chemically-crosslinked conditions will be determined. The molecular weight of a CaCC complex should be some integer multiple of the weight of individual TMEM16A subunits. Lastly, GFP-tagged TMEM16A will be expressed in oocytes and photobleached under TIRF microscopy. The number of fluorophore bleaching events in a single CaCC punctum should equal the number of TMEM16A subunits present. A second set of experiments in this research plan will identify the protein domains responsible for subunit oligomerization. Systematic mutations will be made in TMEM16A's open reading frame and co- immunopreciptation and electrophysiological experiments will determine which mutations abolish subunit interactions. From this data, homologous domains in TMEM16F (a TMEM16 family member that does not normally co-immunoprecipitate with TMEM16A) will be replaced with TMEM16A sequences to determine whether these sequences are sufficient for chimeric proteins to co-immunoprecipitate with wildtype TMEM16A. PUBLIC HEALTH RELEVANCE: The goal of this project is to examine the structure of the mammalian calcium-activated chloride channel. This channel is considered a possible target for the treatment of hypertension, cystic fibrosis, and cancer. As there are currently no specific small-molecule blockers nor activators of this channel, my research will contribute to drug design that will find molecules to efficiently target this ion channel in these diseases.